建立金針菇同源性篩選系統及B型肝炎口服疫苗之開發

Abstract

菇類分子農場(mushroom molecular pharming)係以菇類為生物反應器，應用於醫藥用蛋白質或其他特定蛋白質的生產，具備安全性高、製程簡單和成本低廉的優勢，特別是在口服疫苗的開發。先前已於金針菇建立穩定的農桿菌媒介轉形系統表現異源蛋白質。然而，其實際應用與發展仍然受限於細菌性抗生素篩選標記與低表現量兩大問題。因此，本研究以金針菇單一點突變琥珀酸脫氫酶(succinate dehydrogenase, Sdh)作為同源性篩選標記(homologous selectable marker)，建立非細菌性抗生素篩選系統，提升轉基因菇類的生物安全性。此外，發現刪除金針菇甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase, GPD)啟動子部分序列可明顯提升下游基因表現量。將其應用於功能性蛋白質B型肝炎表面抗原(hepatitis B surface antigen, HBsAg)之表現，產量可達329.08 ± 35.37 ng/g total soluble protein。希望藉由部分刪除之啟動子提升產量與同源性篩選，應用於金針菇B型肝炎口服疫苗之開發展，拓展菇類分子農場未來的應用。

The mushroom molecular pharming refers to the use of mushrooms as bioreactor to produce valuable and pharmaceutical proteins, especially oral vaccines. Previously, a reliable Agrobacterium-mediated transformation for Flammulina velutipes has been established. However, the application was limited due to bacterial antibiotic selectable marker and low yield in heterologous proteins. Until now, the selectable markers using antibiotic resistance genes raise public concerns of biosafety. In this study, we developed a homologous selectable strategy via a single point mutation of succinate dehygrogenase to get transformants against harmless agent, carboxin. Besides, the heterologous gene expression was greatly enhanced by partial deletion of the glyceraldehyde-3-phosphodehydrogenase (gpd) promoters. We also successfully expressed the hepatitis B virus surface antigen (HBsAg) by partial deletion of gpd promoter with a maximum expression of 329.08 ± 35.37 ng/g total soluble protein. We demonstrated that the homologous selectable marker and the enhancement of protein expression using partial deletion of gpd promoter provide useful strategies in development of oral vaccines in F. velutipes.